Synthesis and Biological Evaluation of 4-(Hydroxyalkyl)estradiols and

ChemInform Abstract: Synthesis and Biological Evaluation of 4-(Hydroxyalkyl)estradiols and Related Compounds. C. J. LOVELY , A. S. BHAT , H. D. COUGHE...
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Synthesis and Biological Evaluation of 4-(Hydroxyalkyl)estradiols and Related Compounds Carl J. Lovely,† Abhijit S. Bhat, Holly D. Coughenour, Nancy E. Gilbert, and Robert W. Brueggemeier* Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and the OSU Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210 Received March 13, 1997X

A series of synthetic estrogens containing hydroxyalkyl side chains at the C-4 position of the A ring were designed as metabolically stable analogs of 4-hydroxyestradiol, a catechol estrogen. These synthetic steroids would facilitate investigations on the potential biological role of catechol estrogens and also enable further examination of the structural and electronic constraints on the A ring in the interaction of estrogens with the estrogen receptor. Catechol estrogens are implicated as possible causative agents in estrogen-induced tumorigenesis. 4-Hydroxyestradiol has weaker affinity for the estrogen receptor and exhibits lower estrogenic activity in vivo; on the other hand, the catechol estrogens are prone to further oxidative metabolism and can form reactive intermediates. This report describes the synthesis and initial biochemical evaluation of 4-(hydroxyalkyl)estrogens and 4-(aminoalkyl)estradiols. The 4-(hydroxyalkyl)estrogens were prepared by oxidative hydroboration of 4-alkenylestradiols. The alkenylestradiols were obtained via a Stille cross-coupling between a MOM-protected 4-bromoestradiol and an alkenylstannane. The (4-aminoalkyl)estrogens were prepared from the hydroxyalkyl derivatives with phthalimide under Mitsunobu conditions. The substituted estradiols were evaluated for estrogen receptor binding activity in MCF-7 human mammary carcinoma cells, and 4-(hydroxymethyl)estradiol 1 exhibited the highest affinity with an apparent EC50 value of 364 nM. The relative activities for mRNA induction of the pS2 gene in MCF-7 cell cultures by the 4-(hydroxyalkyl)estrogens closely parallel the relative binding affinities. 4-(Hydroxymethyl)estradiol 1 did not stimulate the growth of MCF-7 cells at concentrations up to 1 µM. Thus, 4-(hydroxymethyl)estradiol 1 exhibited similar estrogen receptor affinity as the catechol estrogen, 4-hydroxyestradiol, and may prove useful in the examination of the biological effects of 4-hydroxyestrogens. Introduction Estrogens are involved in numerous physiological processes including the development and maintenance of the female sexual organs, the reproductive cycle, reproduction, and various neuroendocrine functions. These hormones also have crucial roles in certain disease states, particularly in mammary and endometrial carcinomas. Currently, one out of nine American women will develop breast cancer in her lifetime. Approximately 60% of all breast cancer patients have hormone-dependent breast cancer, with these cancers characterized as containing estrogen receptors and requiring estrogen for tumor growth.1 The possible biochemical roles of estrogens in the development of breast cancer remain to be fully elucidated. Epidemiological studies have shown that women with breast cancer have higher estrogen levels than healthy control women and that estrogen levels are higher in populations characterized by high breast cancer rates.2 An estimated 60-70% of human breast cancers are associated with sex hormone exposure. The fact that an early menarche and a late menopause are important risk factors for breast cancer suggests a role of the female sex hormones in the etiology of the disease.3 Also, studies in experimental animals have shown estrogens to induce tumors in hormone-responsive tissues like * Address all correspondence to: Dr. Robert W. Brueggemeier, College of Pharmacy, 500 West 12th Avenue, The Ohio State University, Columbus, OH 43210. Phone: (614) 292-5231. FAX: 614) 2922435. E-mail: [email protected]. † Present address: Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019-0065. X Abstract published in Advance ACS Abstracts, October 1, 1997.

S0022-2623(97)00168-4 CCC: $14.00

mammary tissue, uterus, cervix, and pituitary.4 Although estrogens have been implicated as carcinogens, the exact biochemical mechanisms by which estrogens may be tumorigenic remain to be established. Catechol estrogens, oxidative metabolites of estrogens, have been suggested as possible causative agents in estrogen-induced tumorigenesis. Estrogens are converted to 2-hydroxy and 4-hydroxy derivatives by cytochrome P-450 hydroxylases.5 Both 2-hydroxyestradiol and 4-hydroxyestradiol have weaker affinity for the estrogen receptor than estradiol and exhibit significantly lower estrogenic activity in vivo.5 However, the catechol estrogens are prone to further oxidative metabolism and can form reactive intermediates like quinones, semiquinones, and arene oxides.6,7 These highly reactive moieties may be cytotoxic via reaction with proteins and nucleic acids.8,9 Furthermore, the catechol estrogens have been shown to produce a variety of reactive oxygen species (ROS), such as the hydroxide, peroxide, and superoxide radicals.10-12 These ROS have shown cytotoxic and genotoxic effects in several independent studies.10,13,14 Contrasting reports exist in the literature in regard to the tumorigenic potential of 2-hydroxyestradiols vs 4-hydroxyestradiols. Liehr et al. recently reported that microsomes prepared from human mammary adenocarcinoma and fibroadenoma have predominantly 4-hydroxylase activity, suggesting a mechanistic role of 4-hydroxyestradiol in tumor formation.15 An earlier report demonstrated that 4-hydroxyestradiol formation is predominant in tissues susceptible to estrogeninduced tumorigenesis like Syrian hamster kidney and © 1997 American Chemical Society

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rat pituitary, whereas 2-hydroxyestradiol formation is predominant in rodent livers where tumors are not produced under similar conditions.16-18 In contrast, Li and Trush found that 2-hydroxyestradiol produced oxidative damage and strand breaks of double-stranded DNA in the presence of micromolar concentrations of Cu(II), whereas 4-hydroxyestradiol failed to produce any DNA damage.13,19 In order to investigate the role of estrogen metabolites in tumor initiation and progression, we have designed, prepared, and reported on a series of 2-hydroxyalkyl derivatives.20 The receptor binding and gene expression potential of these synthetic analogs closely parallels that of 2-hydroxyestradiol. Additionally, these compounds are not able to undergo oxidative metabolism at the 2-position. As a continuing part of this study, we have now prepared the corresponding 4-(hydroxyalkyl)estradiols 1-3. These compounds were designed to provide 4-hydroxy-substituted estrogens that are not able to undergo further oxidative metabolism. On the other hand, compounds 1-3 do contain hydroxyl groups at the 3- and 4-positions that are available for hydrogen bonding during protein interactions with receptors and/ or enzymes. The 4-(aminoalkyl)estrogens, compounds 4-6, were also synthesized from the hydroxyalkyl derivatives to further elucidate electronic factors at the C-4 position that influence biological activity. Therefore, these analogs may prove useful as chemical probes for differentiating receptor-mediated vs redox-mediated events in estrogen-induced tumorigenesis. The synthesis and initial biochemical evaluation of these 4-hydroxyestradiol metabolite analogs are reported in this paper.

nolithium with DMF.22 Unlike the preparation of 7, wherein yields in excess of 80% were routinely realized, only modest yields of 8 could be obtained. As this synthetic intermediate would be required in large quantities, the homologation of 8 was not considered to be the optimal route available for the preparation of 1-3. Alternatively, the bisMOM-protected 4-bromoestradiol 10 was envisioned to be a suitable partner for a Stille cross-coupling reaction.23 Introduction of an appropriate unsaturated group, vinyl or allyl, would afford the hydroxyethyl and hydroxypropyl derivatives, respectively, after hydroboration and oxidation. The synthesis commenced by brominating estradiol with N-bromosuccinimide in ethanol (Scheme 1), from which the required 4-bromoestradiol 9 precipitated and was obtained in 54% yield after recrystallization. The bromoestradiol was protected in 75% yield as its bisMOM ether 10 with chloromethyl methyl ether, diisopropylethylamine in THF at reflux.20 Using vinyltributyltin as the alkenyl donor, exploratory experiments were performed to determine the optimal reaction conditions required for the cross-coupling reaction. Thus, reaction of 10 with tetrakis(triphenylphosphine)palladium(0) (0.06 molar equiv) and vinyltributyltin (2.1 molar equiv) in dry deoxygenated DMF afforded the desired 4-vinyl-bisMOM-estradiol 11 in 90% yield after heating at reflux overnight. Under similar reaction conditions, 10 was treated with allyltributyltin, affording 4-allyl-bisMOM-estradiol 12 in 94% yield. Using well-established chemistry, the unsaturated estradiols 11 and 12 were converted into alcohols 2 and 3. Thus, hydroboration of 11 with BH3‚THF, followed by oxidative workup of the alkylborane with basic hydrogen peroxide, gave the desired alcohol 13. The allylestradiol 12 was transformed into 14 in a similar fashion in 75% yield. Subsequent treatment of alcohols 13 or 14 with pyridinium p-toluenesulfonate (PPTS) gave the targeted triols in 70% and 61% yields, respectively. An attempt was made to prepare the 4-formylestradiol 8 by way of a Stille-like reductive carbonylation as a prelude to preparing alcohol 1. Treatment of 10 with carbon monoxide, tributyltin hydride, and tetrakis(triphenylphosphine)palladium(0) in DMF at reflux failed to yield 8. A control reaction in which 8, prepared by the Pert and Ridley method, was heated for several hours in refluxing DMF demonstrated that it was thermally labile.22 Indeed, a sample of 8 deteriorated simply on standing at room temperature for a few days. In view of the instability of 8, alternate routes for the preparation of other related derivatives were developed. Attempts have been made by Pert and Ridley to introduce an ester group by trapping the organolithium, generated from 10 and n-BuLi with alkyl chloroformates; these reactions were unsuccessful. Treatment of 10 with organolithium (vide supra) and carbon dioxide, followed by acidification and subsequent esterification with diazomethane, yielded the methyl ester 15 in 76% yield (Scheme 2). The MOM protecting groups were removed using PPTS in methanol at reflux in 88% yield. Subsequent reduction of the ester 16 with lithium aluminum hydride gave the benzyl alcohol 1 in 51% yield. The preparation of the 4-substituted amines was accomplished using chemistry similar to that employed for the 2-substituted analogs previously reported.20,21

Results and Discussion Chemistry. In our earlier work, the 2-(hydroxyalkyl)estradiols were prepared via homologation of a protected 2-formylestradiol 7.20,21 Pert and Ridley have previously demonstrated that the analogous 4-formylestradiol 8 could be prepared from 10 by lithiumhalogen exchange and subsequent reaction of the orga-

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Scheme 1a

a Reagents and conditions: (a) N-bromosuccinimide, EtOH, 54%; (b) MOMCl, i-Pr NEt, THF, ∆, 75%; (c) Pd(PPh ) , CH dCHSnBu , 2 3 4 2 3 DMF, ∆, 90%; (d) Pd(PPh3)4, CH3CHdCHSnBu3, DMF, ∆, 94%; (e) (i) BH3‚THF, THF, 0 °C, (ii) NaOH, H2O2, ∆, 11 f 13 39%, 12 f 14, 82%; (f) PPTS, MeOH, ∆, 13 f 2 80%, 14 f 3 61%.

Scheme 2a

a Reagents and conditions: (a) (i) n-BuLi, THF, -78 °C, (ii) CO , 2 -78 °C f rt; (iii) CH2N2, Et2O, 0 °C, 76%; (b) PPTS, MeOH, ∆, 83%; (c) LiAlH4, THF, 0 °C f rt, 51%.

Treatment of the bisMOM-protected 4-(hydroxylalkyl)estradiols (13, 14, 17) with phthalimide under Mitsunobu conditions using triphenylphosphine (PPh3) and

diethyl azodicarboxylate (DEAD) yielded derivatives 18-20 in 70-80% yield (Scheme 3). Subsequent hydrazinolysis in refluxing ethanol gave the bisMOMprotected aminoestradiols which, upon treatment with methanolic HCl, gave the desired 4-(aminoalkyl)estradiols 4-6 in good yields. Biology The affinities of the synthetic 4-hydroxyestradiol analogs for the estrogen receptor were assessed in whole cell estrogen receptor binding assays using MCF-7 human mammary cancer cells.20 The whole cell binding assay provides similar relative binding affinities (RBAs) for the estrogen receptor as those obtained using isolated estrogen receptor preparations.20 In addition, the cellular uptake and stability of analogs in the whole cell assay can be assessed. The EC50 value for estradiol binding to the estrogen receptor in these whole cell assays was found to be 0.180 nM (Table 1). The synthetic hydroxyestrogen analog with the highest estrogen receptor affinity was 4-(hydroxymethyl)estradiol 1, exhibiting an EC50 value of 364 nM. Overall, the 4-substituted estradiol homologs exhibited significantly weaker affinity for the estrogen receptor than estradiol,

Scheme 3a

a

Reagents and conditions: (a) PhthNH, DEAD, Ph3P, THF; (b) NH2NH2, EtOH, ∆; (c) HCl, MeOH.

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Table 1. Estrogen Receptor Affinity of 4-Substituted Estradiol Analogs steroid

compd

EC50 (M)

log EC50 ( SD

RBA

1 2 3 4 5 6

1.80 × 10-10 5.06 × 10-7 3.64 × 10-7 6.20 × 10-7 3.32 × 10-6 NBa 2.50 × 10-6 NB

-9.744 ( 0.102 -6.295 ( 0.092 -6.438 ( 0.141 -6.207 ( 0.290 -5.479 ( 0.116

100.00 0.36 0.49 0.29 0.05

-5.600 ( 0.159

0.07

estradiol 4-hydroxyestradiol 4-(hydroxymethyl)estradiol 4-(hydroxyethyl)estradiol 4-(hydroxypropyl)estradiol 4-(aminomethyl)estradiol 4-(aminoethyl)estradiol 4-(aminopropyl)estradiol a

NB ) no measurable binding of steroid at 10-5 M concentration.

Table 2. Induction of pS2 Gene Expression by 4-Substituted Estradiol Analogs steroid estradiol 4-hydroxyestradiol 4-(hydroxymethyl)estradiol 4-(hydroxyethyl)estradiol 4-(hydroxypropyl)estradiol

compd

EC50 (M)

log EC50 ( SD

% relative activity

1 2 3

3.01 × 10-11 6.54 × 10-8 1.17 × 10-8 1.48 × 10-7 2.95 × 10-6

-10.520 ( 0.217 -7.184 ( 0.158 -7.933 ( 0.288 -6.829 ( 0.094 -5.530 ( 0.217

100.00 0.046 0.257 0.020 0.001

Figure 1. Estrogen receptor competitive binding assays for estradiol (9), 4-hydroxy-methylestradiol (b), and 4-hydroxypropylestradiol (0).

with relative binding affinities (RBA; estradiol ) 100) ranging from 0.49 for compound 1 to 0.05 for compound 3 (Table 1, Figure 1). The relative estrogenic activities of the 4-hydroxyestradiol analogs were evaluated by examining the abilities of the synthetic compounds to induce estrogendependent gene expression in human breast cancer cells. In human MCF-7 mammary carcinoma cells, the induction of transcription of the pS2 gene is a primary response to estrogen.24 The induction of pS2 mRNA expression by estradiol, 4-hydroxyestradiol, and 4-(hydroxyalkyl)estrogen analogs 1-3 was determined by RNA dot blot analysis. The EC50 value for estradiol induction of pS2 mRNA was found to be 0.030 nM. The estradiol homologs exhibited activity significantly weaker than that of estradiol for pS2 mRNA induction, with relative activities (estradiol ) 100) ranging from 0.257 for compound 1 to 0.001 for compound 3 (Table 2, Figure 2). The effects of 4-(hydroxymethyl)estradiol 1 on the growth of hormone-dependent MCF-7 breast cancer cells was investigated since the compound exhibited the highest estrogen receptor affinity of the 4-hydroxylalkyl analogs synthesized. This mitogenic activity was determined by measuring [3H]thymidine incorporation25 at day 4 at synthetic estrogen concentrations ranging from 2.5 to 10 µM. 4-(Hydroxymethyl)estradiol did not affect cellular DNA synthesis in this breast cancer cell

Figure 2. Induction of pS2 gene expression by estradiol (b), 4-hydroxyestradiol (2), 4-(hydroxymethyl)estradiol (0), 4-(hydroxyethyl)estradiol (3), and 4-(hydroxypropyl)estradiol ([).

Figure 3. Comparison of mitogenic activities of estradiol (black bar), 4-(hydroxymethyl)estradiol (gray bar), and vehicle control (white bar) in MCF-7 human mammary carcinoma cell cultures.

line, whereas estradiol at a concentration of 1 nM significantly increased MCF-7 cell growth (Figure 3). Conclusions The Stille cross-coupling and the carboxymethylation reaction reported here represent two efficient, previously unexplored synthetic routes for the functionalization of the 4-position of estradiol. The synthesis of the 4-(hydroxyalkyl)estrogens was accomplished by oxida-

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tive hydroboration of 4-alkenylestradiols, which were obtained via a Stille cross-coupling between a MOMprotected 4-bromoestradiol and an alkenylstannane. The 4-(aminoalkyl)estrogens were prepared from the hydroxyalkyl derivatives with phthalimide under Mitsunobu conditions. The substituted estradiols were evaluated for estrogen receptor binding activity using whole cell receptor binding assays in MCF-7 human mammary cell cultures. The 4-(hydroxyalkyl)estradiols had significantly lower affinity for the estrogen receptor when compared with the endogenous ligand, estradiol. 4-(Hydroxymethyl)estradiol (1) exhibited the highest affinity of the synthetic compounds, with an apparent EC50 value of 364 nM, and it exhibited an affinity similar to that of the endogenous metabolite, 4-hydroxyestradiol, in the whole cell assays. On the other hand, the 4-(aminoalkyl)estradiols (4-6) exhibited either extremely weak or no affinity for the estrogen receptor. Estradiol acts through the nuclear estrogen receptor to induce the transcription of a variety of hormoneresponsive genes in target tissues, and induction of pS2 gene transcription is a primary response to estrogen observed in human MCF-7 mammary carcinoma cells.22 The 4-(hydroxyalkyl)estradiols had significantly decreased efficacy for the induction of pS2 mRNA levels in MCF-7 cells when compared with the endogenous ligand, estradiol. Again, 4-(hydroxymethyl)estradiol (1) was the most potent among the synthetic compounds, with an apparent EC50 value of 11.7 nM. This synthetic compound was more effective than the endogenous metabolite, 4-hydroxyestradiol, which exhibited an apparent EC50 value of 65.4 nM. Thus, the 4-(hydroxyalkyl)estradiols 1-3 exhibited both significantly weaker estrogen receptor affinities and abilities to induce pS2 gene expression in MCF-7 cell cultures. These results are consistent with the established structure-activity relationships of estrogens and the limitations of A ring substitutions on the estrogen molecule in producing estrogen receptor-mediated responses. On the other hand, 4-(hydroxymethyl)estradiol (1) exhibited similar estrogen receptor affinity and similar induction of pS2 gene transcription as the catechol estrogen, 4-hydroxyestradiol. This catechol estradiol has been implicated as a possible causative agent in estrogen-induced tumorigenesis; however, in vitro and in vivo investigations with 4-hydroxyestradiol are difficult due to its chemical and biochemical instability. Thus, 4-(hydroxymethyl)estradiol (1) may be viewed as a chemically stable catechol estrogen homolog and may therefore prove useful in examination of the role of catechol estrogens in normal physiology and in pathological states, such as estrogen-induced tumorigenesis.

of hexanes and ethyl acetate. Melting points were determined in open capillaries on a Thomas-Hoover capillary melting point apparatus and are uncorrected. IR spectra were recorded on a Laser Precision Analytical RFX-40 FTIR spectrometer in the phase indicated. 1H NMR and 13C NMR were recorded on an IBM AF/250 spectrometer at 250 and 67.5 MHz, respectively, in CDCl3 solutions unless otherwise indicated using the residual protiosolvent signal as internal reference. Mass spectra were obtained at The Ohio State University Chemical Instrumentation Center on either a VG 70-2505, a Nicolet FTMS-200 or a Finnigan MAT-900 mass spectrometer. Elemental analyses were performed by Oneida Research Services, Inc. (Whitesboro, NY). 4-Bromoestra-1,3,5(10)-triene-3,17β-diol 3,17β-Bis(methoxymethoxy) Ether (10). MOMCl (5.7 mL, 75 mmol) was added dropwise to a cold (0 °C) solution of 4-bromoestradiol (5.26 g, 15.0 mmol) and diisopropylethylamine (21.3 mL, 89.3 mmol) in THF (125 mL). On completion of the addition, the reaction mixture was allowed to warm up to room temperature, stirred for 1 h at the same temperature, and then heated at reflux overnight. The mixture was allowed to cool, and then saturated NH4Cl solution (100 mL) was added. The mixture was extracted with EtOAc (4 × 100 mL), and the combined organic solutions were washed with saturated aqueous brine (100 mL), dried (MgSO4), and concentrated. The crude product was purified by flash column chromatography (SiO2, hexane/ethyl acetate, 9:1) to afford a pale yellow solid, which was recrystallized from hexane to give 4.78 g (72%) of the desired compound as a colorless solid: mp 88-89 °C (lit. mp 97-98 °C); IR (KBr, cm-1) 2925-2785, 1597, 1578, 1473, 1452, 1444, 1402, 1385, 1306, 1261, 1234, 1224, 1205, 1176, 1155, 1122, 1107, 1088, 980, 914, 897, 856; 1H NMR 7.20 (1H, d, J ) 8.6 Hz), 6.95 (1H, d, J ) 8.6 Hz), 5.22 (2H, s), 4.69 (2H, AB q, J ) 6.6 Hz, ∆v ) 3.5 Hz), 3.61 (1H, t, J ) 8.4 Hz), 3.51 (3H, s), 3.37 (3H,s), 2.98 (1H, dd, J ) 5.4, 17.9 Hz), 2.76-2.65 (1H, m), 2.31-2.11 (2H, m), 0.79 (3H, s); 13C NMR 151.8, 137.8, 136.5, 124.8, 116.4, 113.5, 96.1, 95.4, 86.7, 56.2, 55.1, 50.1, 44.3, 43.0, 37.9, 37.3, 31.3, 28.2, 27.4, 26.6, 23.1, 11.7; MS m/z (M+) calcd 440.1341, obsd 440.1388. 4-Ethenylestra-1,3,5(10)-triene-3,17β-diol 3,17β-Bis(methoxymethoxy) Ether (11). A solution of 11 (440 mg, 1.0 mmol), vinyltributyltin (0.62 g, 2.0 mmol), and Pd(PPh3)4 (67 mg, 0.06 mmol) in DMF (15 mL) was deoxygenated by bubbling argon through it for 15 min. The solution was heated at reflux overnight, cooled to room temperature and diluted with ether (50 mL), washed with 5% NH4OH (15 mL), water (4 × 20 mL), and brine (3 × 20 mL), dried (MgSO4), and concentrated. The residue was purified by column chromatography (SiO2, 4:1 hexane/ethyl acetate) to yield 344 mg (90%) of the title compound as a colorless oil, which solidified on standing to a colorless waxy solid: mp 55 °C; IR (KBr, cm-1) 2930, 2888, 2847, 2023, 1698, 1586, 1476, 1444, 1158, 1112, 1055, 1045, 927; 1H NMR 7.16 (1H, d, J ) 8.7 Hz), 6.94 (1H, d, J ) 8.9 Hz), 6.40 (1H, dd, J ) 11.7, 17.9 Hz), 5.55 (1H, dd, J ) 2.3, 17.9 Hz), 5.52 (1H, dd, J ) 2.3, 11.7 Hz), 5.12 (2H, s), 4.64 (2H, s), 3.60 (1H, t, J ) 8.3 Hz), 3.45 (3H, s), 3.36 (3H, s), 2.91-2.65 (2H, m), 0.74 (3H.s); 13C NMR 152.9, 136.2, 134.4, 131.3, 127.1, 125.0, 119.7, 112.7, 96.1, 95.0, 86.7, 56.0, 55.1, 10.3, 44.5, 43.0, 38.0, 37.5, 28.23, 28.20, 27.4, 26.6, 23.1, 11.7; MS m/z (M+) calcd 386.2457, obsd 386.2443. Anal. (C24H34O4) C, H. 4-(2′-Propenyl)estra-1,3,5(10)-triene-3,17β-diol 3,17βBis(methoxymethoxy) Ether (12). A solution of 11 (1.50 g, 3.41 mmol), Pd(PPh3)4 (250 mg, 0.22 mmol) and allyl tri-nbutyl stannane (2.28 g, 6.90 mmol) in DMF (50 mL) was deoxygenated by bubbling argon through it for 15 min, and then the solution was heated at reflux overnight. After cooling, the solution was decanted off from the palladium, and the residual precipitated palladium was washed with ethyl acetate. The reaction solution was diluted with ethyl acetate, washed with water (3 × 50 mL), and brine (50 mL), dried (MgSO4), and concentrated. The residue was purified by chromatography (SiO2, hexane/ethyl acetate, 10:1) to give 1.28 g (94%) of the desired allyl compound 14 as a colorless oil: IR (neat, cm-1) 2931, 2850, 2825, 1637, 1481, 1446, 1254, 1225, 1205, 1190, 1151, 1134, 1105, 1082, 1055, 1028, 1007, 918; 1H NMR 7.15

Experimental Methods Synthesis: General Information. Estradiol was purchased from Steraloids (Wilton, NH). All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee) and were used as received unless otherwise indicated. Anhydrous solvents were dried by standard procedures. Amines were stirred over CaH2, distilled, and then stored over KOH pellets. Silica gel TLC plates (60 F254) were purchased from Analtech Inc. (Newark, NE) and visualized with a UV lamp and/or 5% ethanolic phosphomolybdic acid followed by charring. All intermediates were purified by flash column chromatography on silica gel (Merck Kieselgel 60) using the indicated mixtures

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Journal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3761

(1H, d, J ) 8.7 Hz), 6.93 (1H, d, J ) 8.7 Hz), 5.97-5.82 (1H, m), 5.17 (2H, s), 4.97 (1H, s), 4.92 (1H, dd, J ) 1.5, 7.1 Hz), 4.65 (2H, AB q, J ) 6.7, ∆ν ) 2.9 Hz), 3.61 (1H, t, J ) 8.3 Hz), 3.45 (3H, s), 3.42 (2H, t, J ) 6.6 Hz), 3.37 (3H, s), 2.91-2.66 (2H, m), 0.79 (3H, s); 13C NMR 153.1, 136.5, 136.3, 134.3, 126.7, 124.1, 114.4, 111.7, 96.1, 94.8, 86.7, 55.9, 55.1, 50.3, 44.4, 43.0, 38.0, 37.5, 30.2, 28.2, 27.4, 26.7, 26.6, 23.1, 11.7; MS m/z (M+) obsd 400.2613, calcd 400.2618. Anal. (C25H36O4) C, H. 4-(2′-Hydroxyethyl)estra-1,3,5(10)-triene-3,17β-diol 3,17β-Bis(methoxymethoxy) Ether (13). A solution of 1 M BH3‚THF (3.00 mL, 3.00 mmol) was added dropwise to a solution of 12 (286 mg, 0.75 mmol) in THF (6 mL) at 0 °C. On completion of the addition, the cooling bath was removed and the mixture stirred for 1 h; 1 M NaOH (3 mL) was added cautiously, and after the addition of 30% H2O2 (3 mL), the mixture was heated at reflux for 1 h. The mixture was allowed to cool, then ethyl acetate (75 mL) was added, and the organic solution was separated from the aqueous layer. The organics were washed with water (25 mL) and brine (25 mL), dried (MgSO4), and concentrated. The residue was purified by MPLC (SiO2, hexane/ethyl acetate, 2:1) to give 37 mg (12%) of a diastereomeric mixture of partially deprotected secondary alcohols, 92 mg (30%) of a mixture of two diastereomeric secondary alcohols, and 119 mg (39%) of the desired primary alcohol as a colorless oil, which crystallized on standing: mp 81-82 °C; IR (KBr, cm-1) 3496, 3311, 2931, 2870, 2844, 2821, 1595, 1581, 1481, 1404, 1385, 1309, 1255, 1227, 1205, 1190, 1149, 1111, 1096, 1068, 1053, 1009, 912, 814; 1H NMR 7.12 (1H, d, J ) 8.7 Hz), 6.93 (1H, d, J ) 8.7 Hz), 5.18 (2H, s), 4.65 (2H, AB q, J ) 6.6, ∆ν ) 3.3 Hz), 3.78 (2H, t, J ) 7.0 Hz), 3.61 (1H, t, J ) 8.4 Hz), 3.46 (3H, s), 3.36 (3H, s), 2.97 (2H, t, J ) 6.7 Hz), 2.96-2.88 (1H, m), 2.82-2.70 (1H, m), 0.79 (3H, s); 13C NMR 153.6, 136.7, 134.6, 125.4, 124.4, 111.5, 96.1, 94.7, 86.7, 62.2, 56.0, 55.1, 50.3, 44.4, 43.0, 37.9, 37.4, 29.5, 28.2, 27.4, 27.1, 26.6, 23.1, 11.7; MS m/z (M+) calcd 404.2563, obsd 404.2574. Anal. (C24H36O5) C, H. 4-(3′-Hydroxypropyl)estra-1,3,5(10)-triene-3,17β-diol 3,17β-Bis(methoxymethoxy) Ether (14). A solution of 1 M BH3‚THF (12.4 mL, 12.4 mmol) was added dropwise to a solution of 14 (1.23 g, 3.08 mmol) in THF (25 mL) at 0 °C. On completion of the addition, the mixture was allowed to warm to room temperature and then stirred for 1 h. NaOH (1 M, 25 mL) was added cautiously, then 30% H2O2 (25mL) was added, and the resulting mixture was heated at reflux for 1 h. The aqueous reaction mixture was extracted with ethyl acetate (3 × 100 mL), and the organic layer was washed with water (100 mL), brine (100 mL) and dried (MgSO4), and concentrated. Column chromatography (SiO2, hexane/ethyl acetate, 2:1) of the residue gave 1.06 g (82%) of the desired alcohol as a colorless oil: IR (neat, cm-1) 3442, 2927, 1479, 1254, 1205, 1151, 1105, 1053, 1024, 920; 1H NMR 7.13 (1H, d, J ) 8.7 Hz), 6.93 (1H, d, J ) 8.7 Hz), 5.18 (2H, s), 4.65 (2H, AB q, J ) 7.0, ∆v ) 0 Hz), 3.67-3.37 (3H, m), 3.48 (3H, s), 3.37 (3H, m), 0.79 (3H, s), 13C NMR 153.3, 136.2, 134.7, 128.6, 123.8, 111.5, 96.1, 95.0, 86.7, 62.4, 86.1, 55.1, 50.3, 44.4, 43.0, 38.0, 37.4, 32.0, 28.2, 27.4, 26.5, 23.1, 21.8, 11.7; MS m/z (M+) calcd 418.2719, obsd 418.2712. Anal. (C25H38O5) C, H. 4-(2′-Hydroxyethyl)estra-1,3,5(10)-triene-3,17β-diol (2). A solution of 13 (80 mg, 0.20 mmol) and pyridinium ptoluenesufonate (0.50 g, 2.00 mmol) in methanol (5 mL) was heated at reflux for 24 h. After the mixture as cooled to room temperature, ethyl acetate (50 mL) was added, and then the solution was washed with water (2 × 50 mL) and brine (50 mL), dried (MgSO4), and concentrated. The residue was recrystallized from methanol and water to afford 50 mg (80%) of the alcohol: mp 229-230 °C; IR (KBr, cm-1) 3338, 29662860, 1591, 1481, 1469, 1444, 1425, 1377, 1358, 1340, 1277, 1200, 1180, 1134. 1072, 1057, 1039, 1011, 818, 810; 1H NMR (DMSO) 8.89 (1H, s), 6.92 (1H, d, J ) 8.48 Hz), 6.56 (1H, d, J ) 8.4 Hz), 4.62 (1H, br), 4.47 (1H, d, J ) 4.8 Hz), 3.54-3.41 (1H, m), 2.84-2.59 (4H, m), 0.63 (3H, s); 13C NMR 151.9, 135.5, 130.7, 123.2, 122.7, 112.1, 79.9, 59.9, 49.5, 43.7, 42.5, 37.8, 36.5, 29.8, 29.4, 27.0, 26.1, 22.6, 11.0; (M+) calcd 316.2038, obsd 316.2032. Anal. (C20H28O3‚0.5H2O) C, H. 4-(3′-Hydroxypropyl)estra-1,3,5(10)-triene-3,17β-diol (3). A solution of the alcohol 14 (173 mg, 0.41 mmol) and pyri-

dinium p-toluenesulfonate (0.50 g, 2.0 mmol) in methanol (5 mL) was heated at reflux for 24 h. After cooling, the reaction mixture was diluted with ethyl acetate (50 mL), and then it was washed with water (3 × 25 mL) and brine (25 mL), dried, and concentrated. The residue was recrystallized from methanol/water to give 61 mg (61%) of the desired alcohol as a colorless solid: mp 240-242 °C; IR (KBr, cm-1) 3392, 3249, 2971, 2916, 2864, 1591, 1491, 1471, 1446, 1425, 1379, 1362, 1280, 1080, 1059, 1034, 1003, 814, 808; 1H NMR (DMSO) 10.28 (1H, s), 6.90 (1H, d, J ) 8.5 Hz), 6.56 (1H, d, 8.5 Hz), 4.47 (1H, d, J ) 4.8 Hz), 4.41 (1H, t, J ) 5.2 Hz), 3.54-3.36 (2H, m), 2.80-2.45 (5H, m), 2.22-2.12 (1H, m), 2.09-2.00 (1H, m), 1.84-1.80 (3H, m), 1.59-1.43 (3H, m), 1.39-1.04 (7H, m), 0.54 (3H, s); 13C NMR 152.6, 134.9, 130.8, 125.9, 122.8, 112.1, 79.9, 60.9, 49.6, 43.8, 42.6, 37.9, 36.5, 31.8, 27.0, 26.2, 25.9, 22.6, 21.8, 11.1; MS m/z (M+) calcd 330.2195, obsd 330.2192. Anal. (C21H30O3‚0.25H2O) C, H. 4-Carboxy-3,17β-Bis(methoxymethoxy)estra-1,3,5(10)triene-3,17β-diol Methyl Ester (15). n-BuLi (3.5 mL, 5.65 mmol) was added dropwise to a solution of 10 (1.15 g, 2.61 mmol) in THF (50 mL) at -78 °C. After 1 h of stirring at this temperature, several pieces of dry ice were added, and then the cooling bath was removed. After being warmed to room temperature, the reaction mixture was diluted with ether (50 mL) and then extracted with 5% KOH (5 × 50 mL). The combined basic extracts were acidified to pH 5 with concentrated HCl and then extracted with ether (6 × 50 mL). The combined ethereal extracts were washed with brine (50 mL), dried (MgSO4), and concentrated to give the crude acid. The acid was suspended in ether (20 mL), and an ethereal solution of diazomethane was added to it at 0 °C. After 30 min, sufficient acetic acid was added to the reaction mixture to discharge the yellow coloration. The reaction mixture was washed with NaHCO3 (2 × 50 mL) and brine (50 mL), dried (MgSO4), concentrated, and purified by chromatography (SiO2, hexane/ethyl acetate, 4:1) to give 0.82 g (76%) of the desired product as a colorless oil, which crystallized after a few days of standing at room temperature: mp 66-68 °C; IR (KBr, cm-1) 2924, 2862, 1728, 1585, 1481, 1440, 1384, 1273, 1257, 1248, 1155, 1126, 1103, 1061, 1049, 1031, 793; 1H NMR 7.25 (1H, d, J ) 8.7 Hz), 6.93 (1H, d, J ) 8.7 Hz), 5.13 (2H, s), 4.64 (2H, AB q, J ) 7.0, ∆v ) 0 Hz), 3.88 (3H, s), 3.59 (1H, t, J ) 8.4 Hz), 3.44 (3H, s), 3.36 (3H, s), 2.79-2.73 (2H, m), 2.291.99 (4H, m), 1.95-1.83 (1H, m), 0.79 (3H, s); 13C NMR 168.9, 151.6, 134.7, 134.6, 127.4, 124.8, 112.6, 96.2, 95.0, 86.7, 56.0, 55.1, 51.9, 50.1, 44.1, 43.0, 38.1, 37.3, 28.2, 26.8, 26.6, 26.4, 23.1, 11.7; MS m/z (M+) calcd 418.2355, found 418.2337. Anal. (C24H34O6) C, H. 4-Carboxy-3,17β-estra-1,3,5(10)-triene-3,17β-diol Methyl Ester (16). A solution of the ester 15 (0.44 g, 1.10 mmol) and pyridinium p-toluenesulfonate (2.77 g, 11.0 mmol) in MeOH (10 mL) was heated to reflux for 36 h. After cooling and addition of EtOAc (100 mL), the organic solution was washed with water (2 × 50 mL) and brine (50 mL), dried (MgSO4), and concentrated. The residue was filtered through a short pad of silica gel (ethyl acetate/hexane, 1:2) to give 0.30 g (83%) of 16 as a colorless oil, which crystallized on standing: mp 135-136 °C (MeOH/H2O); IR (KBr, cm-1) 3435, 2920, 2866, 1718, 1591, 1446, 1427, 1383, 1361, 1344, 1288, 1267, 1230, 1217, 1190, 1171, 1136, 1122, 1059, 1039, 1011, 960; 1H NMR 10.72 (1H, s), 7.38 (1H, d, J ) 8.8 Hz), 6.80 (1H, d, J ) 8.8 Hz), 3.93 (3H, s), 3.72 (1H, t, J ) 8.4 Hz), 3.10-3.04 (2H, m), 2.29-2.06 (3H, m), 1.97-1.84 (2H, m), 0.77 (3H, s); 13C NMR 172.0, 160.0, 139.3, 132.4, 132.1, 115.2, 112.7, 81.8, 51.9, 501, 44.7, 43.3, 37.9, 36.9, 30.7, 29.8, 27.3, 26.9, 23.0, 11.1; MS m/z (M+) calcd 330.1831, found 330.1835. Anal. (C20H26O4‚ 0.5H2O) C, H. 4-Hydroxymethylestra-1,3,5(10)-triene-3,17β-diol (1). LiAlH4 (70 mg, 1.89 mmol) was added portionwise to a solution of the ester 16 (51 mg, 0.15 mmol) in THF (5 mL) at 0 °C. On completion of the addition, the mixture was allowed to warm to room temperature and then stirred for 4 h. When the reaction was complete, water (0.07 mL), 15% NaOH (0.07 mL), and water (0.21 mL) were added. Once a granular precipitate had formed, it was removed by suction filtration through Celite, washed with MeOH, and concentrated to yield 23 mg

3762

Journal of Medicinal Chemistry, 1997, Vol. 40, No. 23

Lovely et al.

(51%) of the desired alcohol as a colorless solid: mp > 270 °C; IR (KBr, cm-1) 3375, 3240, 2960, 2931, 2920, 2866, 2850, 1591, 1479, 1448, 1429, 1383, 1352, 1286, 1252, 1078, 1065, 1009, 820 1H NMR 9.01 (1H, s), 6.99 (1H, d, J ) 8.5 Hz), 6.58 (1H, d, J ) 8.5 Hz), 4.53 (1H, m), 4.48 (2H, AB q, J ) 4.8, ∆v ) 7.7 Hz), 3.55-3.50 (1H, m), 2.98-2.80 (1H, m), 2.78-2.63 (1H, m), 2.30-2.05 (1H, m), 2.03-1.86 (1H, m), 1.85-1.63 (3H, m), 0.67 (3H, s); 13C NMR 153.1, 136.3, 130.7, 124.7, 124.4, 112.7, 79.9, 54.7, 49.5, 43.7, 42.6, 37.9, 36.5, 29.8, 26.8, 26.2, 25.4, 22.6, 11.0; MS m/z (M+) calcd 302.1875, obsd 302.1883. Anal. (C19H26O3‚0.25H2O) C, H. 4-(Hydroxymethyl)-3,17β-bis(methoxymethoxy)estra1,3,5(10)-triene (17). LiAlH4 (340 mg, 9.19 mmol) was added portionwise to a solution of 15 (0.50 g, 1.20 mmol) in THF (30 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature over 1 h and then stirred for 4 h. Water (0.34 mL), 15% NaOH (0.34 mL), and water (1.00 mL) were added successively, and then the resulting grannular precipitate was removed by filtration through a pad of Celite and MgSO4 (1:1). After concentration of the filtrate it was chromatographed to give 0.43 g (90%) of the desired alcohol as a colorless oil, which slowly crystallized: mp 80-81 °C; IR (KBr, cm-1) 3479, 2964-2814, 1597, 1583, 1481, 1441, 1400, 1385, 1255, 1242, 1228, 1153, 1107, 1092, 1063, 1045, 1031, 1005, 995, 955, 903; 1H NMR 7.23 (1H, d, J ) 8.7 Hz), 6.94 (1H, d, J ) 8.7 Hz), 5.19 (2H, AB q, J ) 6.7, ∆v ) 3.4 Hz), 4.74 (2H, m), 4.64 (2H, AB q, J ) 6.6, ∆v ) 3.4 Hz), 3.61 (1H, t, J ) 8.3 Hz), 3.48 (3H, s), 3.36 (3H, s), 3.10-2.92 (1H, m), 2.90-2.81 (1H, m), 2.33-1.91 (6H, m), 1.76-1.11 (9H, m), 0.79 (3H, m); 13 C NMR 153.9, 136.8, 135.1, 128.0, 126.2, 112.6, 96.1, 95.5, 86.7, 56.7, 56.3, 55.1, 50.2, 44.4, 43.0, 37.9, 37.4, 28.2, 27.2, 26.6, 26.5, 23.1, 11.7; MS m/z (M+) calcd 390.2397, obsd 390.2391. Anal. (C23H34O5) C, H. Phthalimides 23-24: General Procedure. DEAD (0.42 mL, 2.40 mmol) was added dropwise to a solution of the alcohol 13, 14, or 17 (0.80 mmol), phthalimide (0.35 g, 2.40 mmol), and triphenylphosphine (0.63 g, 2.40 mmol) in THF (10 mL) at room temperature and then stirred overnight. The solvent was removed in vacuo, and then the residue was dissolved in EtOAc (100 mL), washed with 5% aqueous KOH (4 × 50 mL) and brine (50 mL), dried (MgSO4), and concentrated. The residue was purified by chromatography (SiO2, hexane/ethyl acetate, 4:1) to afford the substituted phthalimides 18-20 as colorless or pale yellow oils, which solidified on standing. 4-(Phthalimidoylmethyl)-3,17β-bis(methoxymethoxy)estra-1,3,5(10)-triene (18): 79%; mp 104-106 °C; IR (KBr, cm-1) 2951, 2925, 2868, 1711, 1479, 1396, 1385, 1348, 1255, 1151, 1113, 1092, 1055, 1028, 1009, 993, 962, 949, 918, 723; 1H NMR 7.79-7.74 (2H, m), 7.69-7.64 (2H, m), 7.20 (1H, d, J ) 8.7 Hz), 6.90 (1H, d, J ) 8.7 Hz), 5.12 (2H, s), 4.90 (2H, s), 4.64 (2H, s), 3.60 (1H, t, J ) 8.3 Hz), 3.36 (3H, s), 3.31 (3H, s), 3.20-3.13 (1H, m), 2.97-2.86 (1H, m), 2.29-1.94 (3H, m), 0.78 (3H, s); 13C NMR 167.9, 137.6, 134.2, 133.7, 132.3, 126.0, 123.0, 122.0, 111.4, 96.1, 94.6, 86.7, 55.8, 55.1, 50.3, 44.4, 43.0, 37.8, 37.4, 34.3, 28.2, 27.4, 26.9, 26.5, 23.1, 11.7; MS m/z (M+) calcd 519.2621, obsd 519.2634. Anal. (C32H39NO6) C, H, N. 4-(Phthalimidoylethyl)-3,17β-bis(methoxymethoxy)estra-1,3,5(10)-triene (19): 87%; mp 117-120 °C; IR (KBr, cm-1) 2930, 2880, 1772, 1716, 1505, 1430, 1393, 1360, 1152, 1119, 1109, 1070, 1052, 998, 900, 720; 1H NMR 7.83-7.67 (4H, m), 7.15 (1H, d, J ) 8.68 Hz), 6.90 (1H, d, J ) 8.67 Hz), 5.16 (2H, s), 4.64 (2H, AB q, J ) 6.8, ∆v ) 2.9 Hz), 3.81 (1H, t, J ) 8.2 Hz), 3.48 (3H, s), 3.35 (3H, s), 3.03-2.75 (4H, m), 0.78 (3H, s); 13C NMR 168.1, 153.6, 136.6, 134.4, 133.7, 132.3, 124.9, 124.6, 123.0, 111.2, 96.0, 94.5, 86.6, 56.0, 50.1, 44.3, 42.9, 37.8, 37.3, 36.8, 28.1, 27.3, 26.7, 26.5, 25.3, 23.0, 11.7; MS m/z (M+) calcd 533.2767, obsd 533.2772 Anal. (C31H37NO6) C, H, N. 4-(Phthalimidoylpropyl)-3,17β-bis(methoxymethoxy)estra-1,3,5(10)-triene (25): 93%; mp 134-135 °C; IR (KBr, cm-1) 2955, 2935, 2894, 2786, 1777, 1726, 1485, 1478, 1445, 1394, 1363, 1153, 1122, 1086, 1055, 1040, 922, 720; 1H NMR 7.85-7.80 (2H, m), 7.73-7.67 (2H, m), 7.13 (1H, d, J ) 8.7 Hz), 6.87 (1H, d, J ) 8.7 Hz), 5.09 (2H, s), 4.64 (2H, AB q, J ) 6.9, ∆ν ) 1.7 Hz), 3.78 (2H, t, J ) 7.2 Hz), 3.59 (1H, t, J ) 8.3 Hz), 3.39 (3H, s), 3.36 (3H, s), 2.88-2.64 (4H, m), 0.78 (3H, s); 13C NMR 168.3, 153.0, 135.7, 134.1, 133.7, 132.2, 128.2,

123.7, 123.0, 111.2, 96.0, 94.4, 86.7, 55.8, 55.0, 50.2, 44.3, 42.9, 38.4, 37.8, 37.4, 28.1, 27.4, 26.6, 26.5, 23.3, 23.0, 11.6; MS m/z (M+) calcd 547.1693, obsd 547.2908. Anal. (C33H41NO6) C, H, N. Amines 4-6: General Procedure: A solution of the phthalimides 18-20 (0.46 mmol) and hydrazine (1 mL) in ethanol (10 mL) was heated at reflux for 1 h. After cooling, ethyl acetate (50 mL) was added, and then the mixture was washed with 5% KOH solution (3 × 25 mL) and brine (25 mL), dried (MgSO4), and concentrated. The residue was dissolved in methanol (5 mL) and cooled to 0 °C, and then HCl was bubbled through it for 15 min. The cooling bath was removed, and then stirring was continued for 3 h. The reaction mixture was concentrated and then redissolved in methanol, and the resulting green solution was decolorized with charcoal. After filtration and concentration, the residue was taken up in the minimum amount of methanol, and the product was precipitated out by the addition of ether, affording the amine salts as colorless or yellow solids. 4-(Aminomethyl)estra-1,3,5(10)-triene-3,17β-diol (4): 90%; mp > 270 °C; IR (KBr, cm-1) 3444-2868, 1620, 1591, 1509, 1491, 1473, 1450, 1379, 1352, 1323, 1284, 1261, 1219, 1201, 1188, 1080, 1057, 1007, 945, 814; 1H NMR (DMSO) 8.42 (4H, brs), 7.15 (1H, d, J ) 8.6 Hz), 6.75 (1H, d, J ) 8.6 Hz), 4.48 (1H, brs), 3.89 (1H, s), 3.52 (1H, t, J ) 8.2 Hz), 2.94-2.70 (2H, m), 2.30-2.22 (1H, m), 1.95-1.70 (3H, m), 1.65-1.49), 0.64 (3H, s); 13C NMR 152.9, 135.4, 130.1, 125.5, 116.8, 111.5, 78.8, 48.3, 42.6, 41.5, 36.7, 35.4, 32.9, 28.8, 25.6, 25.2, 24.8, 21.6, 10.0; MS m/z (M+ - HCl) calcd 301.2037, obsd 301.2042. Anal. (C19H28NO2Cl‚H2O) C, H, N. 4-(Aminoethyl)estra-1,3,5(10)-triene 3,17β-diol (5): 87%; mp >270 °C; IR (KBr, cm-1) 3355, 3299, 3059, 2865, 1589, 1471, 1447, 1383, 1362, 1281, 1270, 1142, 1086, 1066, 1020, 943, 809; 1H NMR (DMSO) 8.5 (4H, brs), 6.92 (1H, d, J ) 8.12 Hz), 6.54 (1H, d, J ) 8.03 Hz), 4.49 (1H, brs), 3.52 (1H, t, J ) 8.01 Hz), 2.80-2.55 (4H, m), 0.64 (3H, s); 13C NMR 153.7, 134.9, 131.2, 123.1, 113.0, 79.8, 49.7, 44.9, 42.4, 38.1, 36.2, 29.9, 27.0, 26.3, 26.1, 22.9, 11.3; MS m/z (M+ - HCl) calcd 315.2198, obsd 315.2201. 4-(Aminopropyl)estra-1,3,5(10)-triene-3,17β-diol (6): 86%; mp >270 °C; IR (KBr, cm-1) 3362, 3276, 3056, 3022, 2962, 2925, 2863, 1635, 1589, 1489, 1443, 1280, 1208, 1133, 1058, 813; 1H NMR (DMSO) 9.2 (1H, brs), 7.91 (3H, s), 6.94 (1H, d, J ) 8.49 Hz), 6.62 (1H, d, J ) 8.38 Hz), 4.50 (1H, d, J ) 4.6 Hz), 3.51-3.44 (3H, m), 2.78-2.73 (4H, m), 0.63 (3H, s); 13C NMR 152.6, 134.9, 130.8, 124.4, 123.2, 112.2, 79.8, 49.5, 43.7, 42.5, 40.5, 37.8, 36.4, 29.8, 26.9, 26.3, 26.1, 25.8, 22.6, 22.0, 11.0; MS m/z (M+ - HCl) calcd 329.2355, obsd 329.2354. Biological Evaluations. General Information. [2,4,6,73H]Estradiol (98.4 Ci/mmol, 3H-E ) was purchased from Du2 pont/NEN (Boston, MA) and was used as received. MCF-7 human breast adenocarcinoma cells were obtained from ATCC, and cells were incubated in a humidified CO2 incubator (Forma model 3052) with 5% CO2 atmosphere. A modified Eagle’s minimum essential medium (MEM) supplemented with essential amino acids (1.5×), vitamins (1.5×), nonessential amino acids (2×), and L-glutamine (1×) was obtained from Gibco BRL (Long Island, NY) and was used for maintaining the cells. The sterilized liquid medium was prepared by the OSU Comprehensive Cancer Center by dissolving the powder in water containing sodium chloride (0.487 g/L), pyruvic acid (0.11 g/L), and sodium bicarbonate (1.5 g/L) and the pH adjusted to 6.8. Fetal calf serum was obtained from Gibco BRL. Steroids were removed from heat-inactivated fetal calf serum by two treatments with dextran-coated charcoal at 57 °C. Tissue culture flasks and supplies were obtained from Corning Glass Works (Corning, NY). Biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Radioactive samples were detected with a Beckman LS 6800 scintillation counter using Formula 963 (Dupont/NEN) as the counting solution. Probes for RNA dot blot analysis (pS2:ATCC 57137; 36B4:ATCC 65917) were obtained as purified plasmids from the American Type Culture Collection and amplified by PCR for use in hybridization. Primers used were synthesized by OLIGOS, ETC (Wilsonville, OR) and were as follows:

4-(Hydroyalkyl)estradiols and Related Compounds

Probes were labeled by random priming with Klenow fragment. Analysis of the RNA dot blots were performed on a Molecular Dynamics PhosphoImager SI. Whole Cell Estrogen Receptor Studies.20 MCF-7 cells were maintained in a similar fashion as described above. Cells from 90-100% confluent cultures were harvested by treatment with 0.01% trypsin solution, and the washed cell pellet was divided into 9.4 cm2 wells on a six-well plate at (1.5-2) × 105 cells/well in modified MEM (2-3 mL) containing 10% steroid free fetal calf serum and gentamycin (20 mg/mL). After 1224 h at 37 °C, the culture media was removed and replaced and with serum-free modified MEM media (888 µL) containing insulin (5.0 mg/L), transferrin (5.0 mg/L), glutamine (2 mM), and albumin (2.0 mg/mL). After 48 h, the media was removed, fresh serum-free-modified MEM media added, and the synthetic estrogens 1-6 at various concentrations (3 × 10-5 to 1 × 10-5 M, 100 µL) were added and incubated for 10 min at 37 °C. To determine total binding, [3H]estradiol (3.0 nM, 1.0 µCi) was added, and the plates were then incubated for 1 h at 37 °C. The cells were washed twice with PBS at 4 °C and then 95% ethanol (1 mL) was added, followed by standing for 30 min at room temperature. An aliquot (500 µL) of the ethanol solution was added to Formula 963 and counted on a liquid scintillation counter. The blank samples with no cells and nonspecific binding samples, containing 6 µM unlabeled estradiol, were performed in a comparable manner. Specific binding of [3H]estradiol was calculated by subtracting the nonspecific binding data from total binding data. The apparent EC50 value for each synthetic estrogen analog represents the concentration of analog to produce a half-maximal displacement of specific [3H]estradiol binding and was calculated by a nonlinear regression analysis (GraphPad Prizm, Version 2.0, GraphPad Software Inc., San Diego, CA). pS2 Induction. MCF-7 cells were maintained in a similar fashion as described above. Cells were plated at a concentration of 5.5 × 105 cells/25 cm2 flask. After 2 days of growth, the cells were rinsed with Ca2+, Mg2+ free PBS and placed on defined media for 48 h. Defined media contained DMEM/F12 media (Gibco BRL) supplemented with human albumin (2.0 mg/ml), transferrin (5.0 mg/L), bovine insulin (5.0 mg/L), and l-glutamine (2 mM). After addition of fresh defined media, the cells were dosed with compound (10-10-10-5 M), 10 nM 17β-estradiol (Sigma, St. Louis, MO), or carrier (95% ethanol). Each compound was tested in triplicate. After 24 h, total cellular RNA was isolated by an adaptation of the method of Chomczynski and Sacchi.20 The cells were lysed with a 4 M guanidine isothiocyanate solution, and the lysate was acidified with 3 M sodium acetate, pH 5.2 (1:10 vol). After addition of 3 M NaOAc, pH 5.2 (1:10 vol), RNA was extracted twice using water-saturated phenol:chloroform:isoamyl alcohol (60:24:1) at pH 4.0. A final extraction using an equal volume of chloroform:isoamyl alcohol (25:1) was performed. RNA from the resulting aqueous layer was precipitated with an equal volume of 2-propanol at -20 °C for 1 h. The RNA was pelleted at 15000g for 30 min at 4 °C. The resulting pellet was washed twice with 70% ethanol and once with 95% ethanol. Dried pellets were resuspended in 30 uL of Dnase-, Rnase-free molecular biology grade water (Sigma Chemical Co.). Quantification of RNA in each sample was performed using the absorbance at 260 nm. Dot Blot Analysis. A denaturing solution containing 50% formamide, 7% formaldehyde, and 1× SSPE was added to 15 µg of RNA from each sample. The RNA was denatured at 68 °C for 15 min. Two volumes of 10× SSPE was added to each sample. The samples were loaded onto a 0.45 µm, positively charged, nylon membrane (Schleicher and Schuell, Keene, NH) using gentle suction through a 96-well dot blot manifold (BioRad, Hercules, CA). Membranes probed for pS2 gene expression were loaded with 10 µg RNA, the remaining 5 µg was loaded onto a membrane probed for the control gene, 36B4.

Journal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3763 Membranes were baked at 80 °C for 1 h and then incubated for at least 3 h in a prehydridization solution containing 5× SSPE, 5× Denhardts Reagent, 2% SDS, 100 µg/mL salmon sperm DNA, and 50% formamide. pS2 and 36B4 cDNA was prepared as described above and used to make 32P-radiolabeled probes using random primers in the RadPrime Kit (Gibco BRL). Probes with specific activity ranging from 5.0 × 105 to 2.0 × 106 cpm/ng were used. Membranes, probed separately for pS2 or 36B4, were incubated for 48 h or 24 h, respectively, in hybridization solution containing 5× SSPE, 5× Denhardts reagent, 1% SDS, 100 µg/mL salmon sperm DNA, 10% PEG, and 50% formamide. The membranes were washed in 0.5× SSPE, 60′, at 55 °C; 0.1× SSPE, 60′, 60 °C; and 0.1× SSPE, 60′, 65 °C. Phosphor screens were exposed for at least 1 h and scanned on the PhosphorImager SI (Molecular Dynamics). Quantification of the signal was performed using ImageQuaNT software (Molecular Dynamics). The apparent EC50 value for each synthetic estrogen analog represents the concentration of analog to produce a half-maximal induction of pS2 mRNA and was calculated by a nonlinear regression analysis (GraphPad Prizm, Version 2.0, GraphPad Software Inc., San Diego, CA). Cell Growth Assay. Human mammary carcinoma cell lines were maintained in 75-cm2 plastic flasks at 37 °C in a modified Eagle’s MEM (10 mL) containing 10% fetal calf serum and gentamycin (20 mg/L). For cell growth determinations, the mammary carcinoma cells were divided into 9.4 cm2 wells at approximately 100 000 cells/well in modified MEM (2 mL) containing 10% steroid-free fetal calf serum and gentamycin (20 mg/l). After 2 days, media was changed to serum-freemodified MEM and experiments initiated. To determine dosedependent effects, varying concentrations of 4-(hydroxymethyl)estradiol 1 (3 nM to 10 µM in 5 µL of 95% ethanol) were added and incubated for 4 days. Effects on cell division were measured by the addition of [3H]thymidine (1 µCi/well), followed by incubation for 2 h, cell lysis, and determination of [3H]thymidine incorporation into DNA. Each experiment was carried out in quadriplicates, and test compounds were evaluated in experiments performed at least three different times. Statistical differences between control and treated groups were determined using the Student’s t test.

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